Although there has been extensive work developing microfabrication and microfluidic technology for biomedical applications, bottlenecks remain in moving the technology broadly into biomedical research laboratories. Part of the issue is a lack of familiarity with the capabilities of microfabrication on the part of many biomedical researchers. In addition, many potential research projects have constraints that require extensive customization and multiple design iterations, which may not be achievable with the limited number of commercial products available. In an effort to lower the barriers for applying microfabrication techniques to a wide range of biomedical problems, we have developed an in-house microfabrication capability for making templates for PDMS or hydrogel devices using either a dry-film resist or SU-8. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. We are now able to reliably pattern single and double layer template features with lateral dimensions of less than 2 microns, and with heights ranging from a few microns to a few hundred microns. The ability to pattern multiple layers with different heights on a single template enables the production of devices with substantially greater functionality - as an example, structures that can be used for trapping cells in one compartment while providing continuous fluid delivery through larger adjacent channels. We have developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels. This year we have continued to refine techniques for making and manipulating thin (<200 micrometer) micropatterned PDMS layers for use in multilayer devices and bottomless structures, and have begun incorporating track-etched polymer membranes into multilayer structures for co-culture of different cell types. We also continue to develop protocols for surface modification of PDMS and other polymers, as well as techniques for connecting devices to flow-control instruments, such as syringe pumps and pressure controllers. In addition to the capability for photolithographic patterning of microfluidic templates, we have also explored the use of a programmable razor cutter and pressure-sensitive adhesive to directly make thin film structures with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This is a low-cost and convenient method for several applications, for example the ready fabrication of flow cells with two glass walls, or for providing fluidic confinement over already-functionalized surfaces. This year we also added the capability for finite element modeling of transport in microfabricated structures, developing models for oxygen delivery in a bioreactor with micropillars and for chemokine concentration in a microfluidic hydrogel device. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes, over this past year. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NEI, NIAID, NHLBI, NCI, NICHD, and NIBIB. A few representative examples of new and ongoing projects are given below. 1) A collaborative effort with LSB, NIAID, to study chemotaxis in 3-D collagen matrices, for which we have been developing and refining a microfluidic agarose device compatible with high-resolution fluorescence and two-photon imaging. A mixing tee on a separate platform, together with programmable syringe pump flow control, enables the formation of reproducible time-varying spatial gradients. This year, we have worked on developing a 2-D platform compatible with the existing imaging instrumentation, and have continued to use finite element models and quantitative image analysis to assist in characterization and modeling of the 3-D device for different temporal inputs. 2) The continuing development, in collaboration with LCE, NHLBI, of gratings for phase-contrast x-ray imaging, which involves the use of the NIST nanofabrication facilities as well as our own equipment. This year our efforts have focused on supporting characterization and publication of imaging results from the current generation of gratings. Preliminary work on the next generation of gratings is underway. 3) In collaboration with MDP, NIDDK, the use of coverslip-mounted, bottomless PDMS microwells to confine eggs in order to study fertilization with fast, high-resolution microscopy. For this application, the wells need to be compatible with existing fluorescence imaging and culture systems, and also to enable capture of the eggs with minimal handling. The addition of microchannels connecting the wells has provided space for the cumulus cells as they separate, enabling the eggs to be gently captured and imaged at the coverslip surface. 4) The development, fabrication, and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, laser capture microdissection. This work, begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, is aimed at developing methods for tissue-based capture of subcellular structures for mass spectrometry-based proteomic analysis. This year, efforts focused on assessing the utility and in particular the capture specificity by looking at the level of enrichment of nuclear proteins using quantitative proteomic analysis. In addition, work continues on refining the instrumentation and microdissection protocols, including methods for quantitative analysis of the capture specificity and efficiency. 5) The development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. This year we also began work on the development of customized image analysis software to extract relevant neurite growth parameters from statistically meaningful numbers of cultured cells. 6) The design, fabrication, modeling, and use of a micropillar array to deliver oxygen to three dimensional cell culture volumes with in vivo like spatial distribution, in collaboration with LCB, CCR, NCI. Efforts in our group this year focused on the refinement of device fabrication protocols and on the development of finite element models of oxygen delivery within the microstructured device, supported by experimental verification. 7) The use of a microfluidic channel together with custom instrumentation developed in LCMB, NICHD in order to separate the effects of pressure and shear rapid transients on cultured neural cells. This new project, headed by LCMB, NICHD, is aimed at better understanding the cellular mechanisms of blast-induced traumatic brain injury. 8) In collaboration with the VRC,NIAID, a new project aimed at using microfabricated structures, including wells and droplet generators, together with high-throughput sequencers to acquire data on genetic diversity at the single-cell level.